Method and compositions for treatment or prevention of pneumocystic carinii infections

ABSTRACT

The present invention provides methods for treating and preventing the onset of Pneumocytis carinii pneumonia (PCP) comprising administering to a mammal in need of such treatment or prevention an effective amount of a pharmaceutically acceptable iron chelator. This invention also provides a method for treating or preventing the onset of PCP comprising administering to a mammal in need of such treatment or prevention a composition comprising the following components: (a) a pharmaceutically acceptable iron chelator; (b) a compound selected from trimethoprim/sulfamethoxazole and pentamidine; and (c) a pharmaceutically compatible carrier or diluent, wherein the amounts of said component (a) and said component (b) in combination are effective in treating said pneumonia. Also provided is a composition for the treatment or prevention of PCP.

This is a division of application Ser. No. 07/910,714, filed Jul. 7,1992, now U.S. Pat. No. 5,302,598, which is a division of applicationSer. No. 340,344, filed Apr. 19, 1989 (now U.S. Pat. No. 5,158,979issued Oct. 27, 1992).

BACKGROUND OF THE INVENTION

This invention relates to methods and compositions for treating and/orpreventing Pneumocystis carinii infections in mammals.

P. carinii pneumonitis (PCP) is one of the leading causes of death ofvictims of acquired immunodeficiency syndrome (AIDS). Untreated, themortality rate of PCP in AIDS patients approaches 100%. During 1986, thenumber of deaths from PCP in the United States exceeded the combinednumber of deaths from all types of meningococcal infections, viralhepatitis and encephalitis, gonorrhea, syphilis, varicella, measles,mumps, rubella, diphtheria, tetanus, pertussis, polio, amebiasis,shigellosis, salmonellosis, typhoid fever, typhus fever, cholera,rabies, brucellosis, anthrax, tularemia, botulism, and malaria. Theclinical course of PCP infection in immunosuppressed patients presents anumber of symptoms including tachypnea, cough, fever, hypoxemia (bloodoxygen deficiency), an increased alveolar-capillary oxygen gradient,respiratory acidosis and bilateral diffuse alveolar disease (Hughes, W.T., N. Eng. J. Med., 317: 1021-1023, 1987).

PCP is caused by a ubiquitous lung dwelling organism. Recent evidenceindicates that this pathogen may be a fungus rather than a protozoan aspreviously thought (Meshnick, S. et al., "Antioxidant Enzymes ofPneumocystis carinii", Abstracts from the 36th Annual Meeting of theAmerican Society of Tropical Medicine and Hygiene, p. 235, 1987; Edman,J. C. et al., Nature 334: 519-522, 1988; Stringer et al., "Sequence fromRibosomal RNA from Pneumocystis carinii compared to those of Four FungiSuggests an Ascomycetous Affinity", Journal of Protozoology 36: 14S-16S,1989; Watanabe et al., "5S Ribosomal RNA Sequence of Pneumocystiscarinii and its Phylogenetic Association with)Rhizopoda/Myxomycota/Zygomycota Group", Journal of Protozoology 36:16S-18S, 1989; Edman et al., "Ribosomal RNA Genes of Pneumocystiscarinii", Journal of Protozoology 36: 18S-20S, 1989).

Early potential treatments for the disease were tested on the infantileP. carinii interstitial plasma-cell pneumonitis that occurred inepidemic form in Europe. The mortality rate for the untreated form ofthe affliction was about 50%. At that time, it was determined thatvarious antibiotics and/or anti-microbial agents, such as penicillin,tetracycline, chloramphenicol, streptomycin, quinacrine, chloroquine,neoarsphenamine, stibophen, pamaquine, mulsin, neospiran, arsaphen,arsphenamine, quinine, chloroguanide and emetine hydrochloride wereineffective in treating the disease (Hughes, W., Pneumocystis cariniiPneumonitis, CRC Press Incorporated, 1987).

Defined high risk groups exist for PCP. While the ubiquitous P. cariniiacts as a commensal organism and does not cause disease in a healthyindividual, it may nevertheless produce PCP in, for example, patientsimmunosuppressed due to AIDS, to drugs given for cancer treatment, todrugs given to prevent rejection of organ or tissue transplant, to drugsgiven for treatment of autoimmune disease and other immunocompromised,i.e., partially or totally immunosuppressed or immunodeficient, patientsor hosts. Because PCP poses such a threat to these identifiable highrisk groups, there exists a need for a prophylactic routine as well as atherapeutic treatment.

The development of treatments for PCP has been hindered by the lack ofknowledge about the biology and properties of the P. carinii organism.The nutritional requirements, metabolic pathways, mode of replication,enzyme systems and taxonomy of P. carinii are not well understood.Although there are some newly identified agents active against PCP, onlytwo recognized treatments currently exist for the control of PCP.

The first recognized mode of treatment involves the use of pentamidine,p,p'-(pentamethylenedioxy)dibenzamidinebis(beta-hydroxy-ethanesulfonate) which is an aromatic diamidinocompound. Pentamidine may be prepared according to the disclosure inNewberry Easson, U.S. Pat. No. 2,410,796 and is available commercially,e.g., from LyphoMed, Inc., Rosemont, Ill. 60018).

The second treatment involves the use of a combination of trimethoprimwith sulfamethoxazole (TMP/SMZ), i.e.,5-[(3,4,5-trimethoxyphenyl)methyl]-2,4-pyrimidinediamine/4-amino-N-(5-methyl-3-isoxazolyl)benzenesulfonamide).TMP may be prepared from guanidine andbeta-ethoxy-3,4,5-trimethoxybenzylbenzalnitrile (see, e.g., Stenbuck,Hood, U.S. Pat. No. 3,049,544 and Hoffer, U.S. Pat. No. 3,341,541). SMZmay be prepared starting with ethyl 5-methylisoxazole-3-carbamate (seeKano et al., U.S. Pat. No. 2,888,455). TMP and SMZ, including thecombination of TMP/SMZ, are available commercially from a number ofsources. For example, a suspension of TMP/SMZ (400 mg/200 mg) isavailable from Geneva Generics, Inc., Broomfield, Colo. 80020; in tabletform, TMP/SMZ may be obtained from Par Pharmaceutical, Inc., SpringValley, N.Y. 10977. Other forms of TMP/SMZ-including commercial sourcesmay be found by referring to Physician's Desk Reference, 1988 Edition,Med. Econ. Co., Inc., Oradell, N.J., p.325, col. 2.

The efficacy of the combination of TMP/SMZ derives from the ability ofTMP to inhibit microbial dihydrofolate reductase activity and from thecompetitive interference of SMZ with the incorporation ofpara-aminobenzoic acid into dihydrofolate, which serves to limit theformation of substrate for the enzyme. Known disadvantages of thesetreatments include lack of clinical responsiveness, high rates oftoxicity and numerous other adverse side-effects. In AIDS patients, inparticular, the severe adverse reactions caused by pentamidine therapyinclude neutropenia, thrombocytopenia, rash and alterations in mentalstate, e.g., depression. Symptoms resulting from pentamidine therapyinclude hypoglycemia, hypotension and nephrotoxicity. In addition, ithas been determined that AIDS patients suffering from PCP requiretherapy far longer periods of time and have higher relapse rates(Havertos, H. W., Am. J. Med. 76: 501-508, 1984).

New treatments undergoing clinical trials include various agents withmodes of action similar to that of TMP/SMZ; i.e., interference withfolate metabolism. These include: TMP in combination with dapsone,diaminodiphenylsulfone, a drug used to treat leprosy (Green et al.,"AIDS-Related Pneumocystis carinii Pneumonia Successfully Treated withDapsone-Trimethoprim", British Journal of Clinical Pharmacology 26:487-491, 1988); trimetrexate (a new anti-cancer drug) in combinationwith leucovorin as a rescue agent for host metabolism (Allegra et al.,"Trimetrexate for the Treatment of Pneumocystis carinii Pneumonia inPatients with Acquired Immunodeficiency Syndrome", New England Journalof Medicine 317: 978-985, 1987); high doses of steroids combined withspecific anti-PCP therapy (Gallacher et al., "Treatment of AcutePneumocystis carinii Pneumonia with Corticosteroids in a Patient withAcquired Immunodeficiency Syndrome", CritiCal Care Medicine 17: 104-105,1989); administration of TMP/SMZ with careful monitoring of the serumconcentration in individual patients during treatment so as to minimizeadverse side effects (Sattler et al., "Trimethoprim-SulfamethoxazoleCompared with Pentamidine for Treatment of Pneumocystis cariniiPneumonia in the Acquired Immunodeficiency Syndrome", Annals of InternalMedicine 109: 280-287, 1988). TMP/SMZ has been administeredprophylactically to AIDS patients to prevent PCP similar to the protocolfound to be successful for children undergoing treatment for leukemia.Pentamidine has been formulated as an aerosol for delivery directly tothe lungs primarily as a prophylactic protocol (Kovacs and Masur,"Pneumocystis carinii Pneumonia: Therapy and Prophylaxis", Journal ofInfectious Diseases 158: 254-259, 1988). DL-alphadifluoromethylornithine(DFMO, eflornithine) is based on an entirely new mode of action and isunder clinical evaluation for treatment of PCP (Schechter et al.,"Clinical Aspects of Inhibition of Ornithine Decarboxylase with Emphasison Therapeutic Trials of Eflornithine (DFMO) in Cancer and Protozoandiseases", in Inhibition of Polyamine Metabolism eds: McCann, Pegg andSjoerdsma, Academic Press, pages 345-364, 1987). In addition to the newtreatments described above, several others have been found to be activein animal models of PCP. These include: compounds related to pentamidinesuch as berenil (Clarkson et al., "Efficacy ofDL-alphadifluoromethylornithine in a Rat Model of Pneumocystis cariniiPneumonia", Antimicrobial Agents and Chemotherapy 32: 1158-1163, 1988)and 1,4-di(4'-amidinophenoxy)butane (Tidwell et al., "Treatment ofExperimental Pneumocystis carinii Pneumonia with Analogues ofPentamidine", Journal of Protozoology 36: 74s-77s, 1989); piritrexim,another anti-cancer drug which interferes with folate metabolism and isclosely related to trimetrexate (Queener et al., "Activity ofLipid-Soluble Inhibitors of Dihydrofolate. Reductase againstPneumocystis carinii in Culture and in a Rat Model of Infection",Antimicrobial Agents and Chemotherapy 31: 1323-1327, 1987); and acombination of clindamycin and primaquine (Queener et al., "Activity ofClindamycin and Primaquine against Pneumocystis carinii in vitro and invivo", Antimicrobial Agents and Chemotherapy 32: 807-813, 1988.

It has long been known that in many instances hosts and pathogenscompete for trace nutrients such as iron, and that the ability of apathogen to establish infection is often dependent on the ability of thepathogen to compete successfully against the host for trace nutrients(Jones, R. and Grady, R. W., Eur. J. Clin. Microbiol. 2:411-413, 1983).In order to secure needed iron, many microorganisms produce and releaseextremely effective, low molecular weight iron chelators known assiderophores, which compete with host iron-binding proteins. Themicrobe-produced siderophores allow the pathogens to survive thehypoferemia the host produces in response to infection . The hostincreases the amount of iron-binding proteins such as lactoferrin andtransferrin which sequester iron making it unavailable to the microbe.When the microbe-produced siderophores complex with iron, the complexcan be taken up by the microbe thus satisfying its nutritionalrequirement for iron.

Accordingly, it is known that a low bioavailable iron level maycontribute to reducing susceptibility to certain microorganisms. It hasalso been reported that administration of iron compounds to patientssuffering from certain diseases aggravates the condition (Masawe, A. E.J., et al., Lancet, 2: 314-317, 1974). Therefore, iron chelators, whichact to complex iron, have long been known to have therapeutic potential.One use of iron chelators has been to treat iron overload in patientsreceiving multiple blood transfusions (such as those suffering frombeta-thalassemia). Iron chelators have also been used as antibacterialand antimicrobial agents. For example, hydroxamic acids, a specificclass of iron chelator, have been found to inhibit malaria sporozoites(Hynes, J. B., J. Med. Chem. 13: 1235-1237, 1970). Iron chelators havealso been used in conjunction with antimicrobial agents in the controlof Stapbylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae,Proteus mirabilis, and species of Salmonella, Enterobacter, Pseudomonasand Providencia (van Asbeck, B. et al., Eur. J. Clin. Microbiol. 2:432-438, 1983). Although iron chelators have been employed to treatvarious microbial infections, their use is by no means predictable oruniformly effective. As an example, desferrioxamine, a hydroxamic acidiron chelator, has been shown to increase the virulence of S.typhimurium in mice (Jones, R. and Grady, R. W., supra).

Desferrioxamine (DFO) is known to be useful in the treatment of otherillness; its ability to chelate aluminum has been exploited in thetreatment of Alzheimer's disease (Mclachlan, U.S. Pat. No. 4,419,365issued Dec. 6, 1985). It has also been used to suppress Plasmodiumfalciparum malaria (Pollack, S. et al., Proc. Soc. Exp. Diol. Med. 184:162-164, 1987).

OBJECTS OF THE INVENTION

It is an object of the present invention to provide a method fortreating Pneumocystis carinii pneumonia (PCP) in mammals.

Another object of the present invention is to provide a method fortreating PCP by administering to a mammal in need of such treatmenttherapeutically effective amounts of a pharmaceutically acceptable ironchelator.

Yet another object of the present invention is to provide a method forpreventing the onset of PCP in a mammal in need of such prophylaxis byadministering prophylactically a therapeutically effective amount of apharmaceutically acceptable iron chelator.

A still further object of this invention is to provide a composition lowin toxicity for treating or preventing the onset of PCP in mammals.

These and other objects of the present invention will be apparent tothose of ordinary skill in the art in light of the present descriptionand appended claims.

SUMMARY OF INVENTION

The present invention involves the discovery that iron chelators presentan effective methodology for treating or preventing Pneumocystis cariniipneumonia (PCP) in mammals. The present inventors have found that ironchelators or chelating agents, such as hydroxamic acids, e.g.,desferrioxamine, suppressed PCP in a rat model of the disease withoutthe adverse side effects prevalent in the two current therapies.Additionally, such chelators have a prophylactic effect to which endthey are preferably administered in a slow or in a sustained releasemedium.

The present invention provides a method of treating Pneumocystic cariniipneumonia (PCP) which comprises administering to a mammal in need ofsuch treatment an effective amount of a pharmaceutically acceptable ironchelator.

This invention also provides a method for preventing the onset ofPneumocystis carinii pneumonia (PCP) which comprises administering to amammal in need of such prevention an effective amount of apharmaceutically acceptable iron chelator.

This invention further provides a method of treating or preventing theonset of Pneumocystis carinii pneumonia which comprises administering toa mammal in need of such treatment a composition comprising thefollowing components: (a) a pharmaceutically acceptable iron chelator;(b) a compound selected from trimethoprim/sulfamethoxazole, andpentamidine or compounds with related modes of action; and (c) apharmaceutically compatible carrier or diluent, wherein the amounts ofsaid component (a) and said component. (b) in combination are effectivein treating or preventing said pneumonia.

In one aspect, the present invention is directed to a composition forthe treatment or prevention of Pneumocystis carinii pneumonia in mammalscomprising the following components: (a) a pharmaceutically acceptableiron chelator; (b) a compound selected fromtrimethoprim/sulfamethoxazole and pentamidine; and (c) apharmaceutically compatible carrier or diluent, wherein the amounts ofsaid component (a) and said component (b) in combination are effectivein treating or preventing said pneumonia.

In another aspect, this invention provides a composition for thetreatment or prevention of Pneumocystis carinii pneumonia in mammalscomprising:

(a) an amount of a pharmaceutically acceptable hydroxamic acid;

(b) an amount of pentamidine; and ,

(c) a pharmaceutically compatible carrier; wherein the total amounts ofsaid components (a) and (b) in combination being effective in treatingor preventing said pneumonia.

DETAILED DESCRIPTION OF THE INVENTION

All literature references and patents cited in this specification arehereby incorporated by reference in their entirety.

The present inventors have discovered that iron chelators can be usedeffectively alone, or in combination with other active agents, to treatPneumocystis carinii pneumonia (PCP) in mammals. Previously, ironchelators, specifically desferrioxamine (DFO), had been used safely,i.e., with minimal adverse side effects, in patients, for example withbeta thalassemia, who receive multiple blood transfusions and oftensuffer from iron overload as the result of such repeated transfusions.

As used herein, treatment is defined as suppression of PCP diseasesymptoms in mammals, e.g., individuals, with clinically apparent diseasedue to P. carinii and/or prevention of the disease in high-riskindividuals. The latter class includes patients immunosuppressed due toAIDS as well as immunocompromised patients, i.e., those who areimmunodeficient or immunosuppressed, e.g., genetically, or throughdisease or drug treatment. Mammals who are at risk to contract PCP alsoinclude mammals having at least partial acquired immunodeficiency andmammals having at least partial congenital immunodeficiency.Immunosuppressed host thus includes patients who through a geneticcondition, disease or drug therapy have impaired, suppressed orinadequately developed immune systems. Specific nonlimiting examples ofsuch patients are those suffering from AIDS or AIDS-Related complex(ARC-a prodrome of the disease), cancer patients undergoing X-ray orchemotherapy treatments and patients with a compromised immune systemsuch as those individuals receiving immunosuppressive therapy becausethey have received or are preparing to receive an organ transplant,patients receiving immunosuppressive therapy for treatment of autoimmunedisease, and individuals with congenital immunodeficiency, e.g.,congenital agammaglobulinemia, Bruton's agammaglobulinemia, andNezelof's syndrome.

The iron chelators of the present invention can be utilized for thetreatment of mammals afflicted with PCP. Due to their high efficacy andlack of serious side effects, the iron chelators of the invention willalso be particularly useful prophylactically for those at high risk ofdeveloping PCP as discussed above.

The iron chelators of the present invention are preferably administeredin water soluble form such as in pharmaceutically acceptable salts oresters. For example, salts are contemplated in which the chelatoractivity resides in the cation or anion and the corresponding ion ispharmacologically suitable. Other forms of iron chelators include thosein which the iron-binding moieties are blocked or modified so as to forma prodrug which on administration to (and/or upon being partially ortotally metabolized by) the host will have iron-chelating activity.

The experiments reported in Examples 1 and 2 below were performed in arat model of PCP. The course of the disease is very similar inchemically immunosuppressed rats and in humans with AIDS. All of theexisting anti-P. carinii therapies were discovered with the aid of therat model and this model is generally considered to be a good predictorof the response in humans.

Broadly, it is known in the art that PCP can be induced byimmunosuppression alone although exposure to other animals withfulminant infection hastens infection in newly immunosuppressed animals.Generally, it takes from 6 months to 1 year to develop an intense,consistent level of infection within a colony of rats in which a portionof the rats are always maintained on an immunosuppressive regimen.

The animals used for the experiments described below were maintained ina room in which a series of other animals had been maintained on animmunosuppression regimen for more than a year and had developed heavyP. carinii infections. In addition, one week after initiation ofimmunosuppression, the rats were injected intratracheally with 0.05 mlof a tissue homogenate made from a rat lung heavily infected with P.carinii. In these experiments, immunosuppression was induced in the ratsby biweekly injection of cortisone acetate. Following the development ofan infection, the rats were subject to various treatment regimens,including maintenance on a low protein diet for the first 14 days ofimmunosuppression and antibiotic administration to protect against otherinfectious agents (see Example 1). In Example 2, the immunosuppressionprotocol was the same as in Example 1 except that the low protein dietwas maintained for the entire experimental period and no amphotericin Bwas administered. Thereafter, the rats were sacrificed and examined forthe existence of established P. carinii infection in the lungs. Thenumber of cysts formed the basis for evaluating the efficacy of thetreatment. Desferrioxamine (DFO) inhibited P. carinii cyst formation 85%in one experiment and over 93% in a second experiment.

In Example 1 below, a large DFO dosage was used, approximately 1g/kg/day. It should be noted, however, that the effective dosage in asmall mammal, e.g., a rodent, is expected to be significantly higherthan that for large mammals, e.g., humans, on a mg/kg basis.Additionally, the administration of DFO to the test animals was by wayof a single daily injection. The preferred administration to humans is aslow intravenous or subcutaneous infusion and such administration wouldresult in a greater effect at a lower dosage. The amount or dosage ofiron chelator to be administered to a mammal in accordance with thepresent invention may be determined by methods known to those skilled inthe art (see, e.g., Jones, R., and Grady, R. W., supra).

The present invention provides a method of treating or preventing theonset of Pneumocystis carinii pneumonia (PCP), which comprisesadministering to a mammal in need of such treatment or prevention aneffective amount of a pharmaceutically acceptable compound orcomposition displaying ironchelating activity in the bloodstream of thehost. Non-limiting examples of suitable iron chelators includearomatic/aliphatic hydroxamic acids such as salicylhydroxamic acid(Aldrich Chemical Co., Milwaukee, Wis.); the mesylate salt ofdesferrioxamine(N-[5-[3-[(5-aminopentyl)hydroxycarbamoyl]propionamido]pentyl]-3-[5-(N-hydroxyacetamido)pentyl]carbamoyl]propionohydroxamicacid monomethane sulfonate (DFO, desferrioxamine, Desferral®), availablefrom CIBA Pharmaceutical Corp., Summit, N.J.; and N'N'-bis(23-dihydroxybenzoyl)-1,6-diaminohexane, described in Bhargava, K. K. etal., J. Pharm. Sci. 69: 986-989, 1980; catechols such as2,3-dihydroxybenzoylglycine described in Ito, T. and Neilands, J. B., J.Amer. Chem. Soc. 80: 4645-4647, 1971; pyridones such as1,2-dimethyl-3-hydroxypyrid -4-one described in Kontoghiorghes, G. J.and Sheppard, L., Inorgan. Chim. Acta 136: L11-12, 1987 and1-methyl-3-hydroxy-pyrid-2-one described in Mohrle, H. and Weber, H.,Tetrahedron 56: 3779-3785, 1970; thiosemicarbazones such as thethiosemicarbazones of picolinaldehyde described in Akiga, S., Japan J.Exp. Med. 26: 91-112, 1956; and 2,5-dihydroxybenzaldehyde, described inLtaniyama, H. et al., J. Pharm. Soc. Japan 76:-1300-1303, 1956; andhydrazones such as pyridoxal isonicotinoyl hydrazone available fromPorphyrin Products, Logan, UT and the guanyl hydrazone of2-acetylpyridine described in Ulrich, P. C. et al., Drug Dev. Res. 2:219-228, 1982. In addition, combinations of any of the foregoing ironchelators (having the property to chelate iron) may be administered inaccordance with the present invention. In general. the iron chelators ofthe present invention bind at least one molecule of iron per molecule ofiron chelator, e.g., DFO, although the iron-abstracting ability willvary with the given chelator. For example, thiosemicarbazones andbis(2,3-dihydroxybenzoyl) glycine (a catechol) bind two molecules ofiron/molecule of chelator. Other iron chelators, such assalicylhydroxamic acid, 1,2-dimethyl-3-hydroxypyrid-4-one, and1-methyl-3-hydroxy-pyrid-2-one bind three molecules of iron/molecule ofchelator while N'N'-bis(2,3-dihydroxybenzoyl)-1,6-diaminohexane bindsthree molecules of iron/two molecules of chelator.

The route of administration depends on the particular chelator. In thecase of desferrioxamine (DFO), parenteral administration is thepreferred route. For example, DFO may be administered to the mammal orpatient by subcutaneous injection. Subcutaneous injection may be carriedout by means of a small portable pump capable of providing continuousinfusion. In most other instances the iron chelator may be orallyadministered. Thus, for example, the following iron chelators are ingeneral, orally administered: hydroxamic acids, e.g., acetohydroxamicacid (used in treatment of gallstones), Bufexamac®(4-butoxy-N-hydroxybenzeneacetamide; 2-(p-butoxyphenyl)acetohydroxamicacid), catechols, e.g., 2,3-dihydroxybenzoylglycine, pyridones, e.g.,1,2-dimethyl-3-hydroxypyrid-4-one, and 1-methyl-3-hydroxy-pyrid-2-one,thiosemicarbazones, e.g., thiosemicarbazones of picolinaldehyde and2,5-dihydroxybenzaldehyde and hydrazones, e.g., pyridoxal isonicotinoylhydrazone.

Those skilled in the art will readily appreciate that drug deliverysystems may be employed in order to adapt a compound, i.e., ironchelator, for a particular route of administration. For example,desferrioxamine (DFO) is generally administered parenterally, e.g.,subcutaneously and to a lesser extent, intravenously. Accordingly, DFOadministration may be combined with pentamidine administration(generally intramuscularly) so that both compounds may be administeredparenterally, e.g., intravenously, to treat or prevent the onset of PCPin a mammal. In addition, by "packaging" DFO in a pro-drug form, forexample, by chemically modifying its structure by esterification, DFOmay be administered orally, then acted upon by the gut and converted toan "active" form after absorption from the gut. Thus, if properlypackaged (chemically modified), DFO could be administered orally incombination with other "oral" iron chelators.

The iron chelators of the present invention may be administered tomammals suffering from PCP in dosages broadly ranging between about 1and about 260 mg/kg body weight per day and preferably between about 5and 100 mg/kg body weight per day. For example, the dosage ofdesferrioxamine mesylate, could preferably range from about 0.2 to about6 g daily, most preferably from about 0.5 to about 2.0 g daily. The ironchelators of the present invention may be administered orally up to 12times daily (where applicable) or as intermittent/continuous,subcutaneous/intravenous infusions lasting 10 min to 24 hours asdetermined by the condition of the patient and stage of disease. Atypical treatment regimen would comprise administration of 25 mg/kg bodyweight, 4 times per day. The duration and number of doses or treatmentsrequired to control a patient's disease will vary from individual toindividual doses of DFO, up to 16 g per day can be tolerated by patientsfor a short course of intensive therapy.

In administering the iron chelator to the mammal or patients in order totreat or prevent the onset of PCP, pharmaceutically compatible carriers,diluents or excipients are employed. Such pharmaceutically compatiblecarriers include by way of example and not limitation, water, includingsterile and deionized water, physiological saline, sodium bicarbonateand glucose. In the case of desferrioxamine (DFO), sterile water is thepreferred pharmaceutically compatible carrier.

In other embodiments of this invention, iron chelators, such asdesferrioxamine (DFO), may be employed in combination with othercompounds previously used in the treatment or prevention of Pneumocystiscarinii pneumonia. Such previously used compounds include but are notlimited to: inhibitors of dihydrofolate reductase such as trimethoprim,methotrexate, trimetrexate, piritrexim and others either singly or incombination with compounds that interfere with the metabolism ofpara-aminobenzoic acid such as sulfamethoxazole, dapsone and othersulfonamides and sulfones, i.e., "sulfa drugs"; polyamine biosynthesisinhibitors such as DL-alpha-difluoromethylornithine, other ornithineanalogues and bis-benzyl polyamines; clindamycin and primaquine, singlyor together; pentamidine and other diamidines such as diminazene(berenil) and 1,4-di(4'-amidinophenoxy)butane; and corticosteroids orother anti-inflammatory or immunosuppressive agents. Such previouslyused compounds include dihydrofolate reductase (DHFR) inhibitingcompounds, i.e., compounds that inhibit the activity or formation ofDHFR, such as trimethoprim/sulfamethoxazole (TMP/SMZ) and pentamidine.In combining iron chelators in compositions with other compounds, thesevere toxic effects of such other compounds may be reduced as aconsequence of employing lesser amounts of such compounds. Accordingly,amounts less than the recognized effective amounts of 20 mg/kg/day and100 mg/kg/day, in the case of TMP and SMZ, respectively, or 4 mg/kg/dayin the case of pentamidine may be employed according to this invention.For use in combination with an appropriate iron chelator, TMP/SMZ may beused over a range of 1 to 20 mg TMP/kg/day and 5 to 100 mg SMZ/kg/dayfor 5 days to 3 weeks, and pentamidine (as the isethionate,methanesulfonate or other salt) over a range of 0.1 to 2.0 mg/kg/day for1 to 14 days to treat or prevent PCP in mammals.

It should be understood that the amounts of component (a) and component(b) in combination which are effective in treating or preventing PCP,also embraces the situation where either component alone, might or mightnot, be effective in treating or preventing the pneumonia. It isexpected that the effect or benefit of using a combination of ironchelator with either TMP/SMZ or pentamidine will at least be additive inthat the mechanisms of action are believed to be different. Accordingly,the effect of the iron chelator should not interfere with the effect ofcomponent (b), i.e., TMP/SMZ or pentamidine, and vice versa.

In another feature of this invention, the iron chelator and/or TMP/SMZ(and/or pentamidine) may be administered in combination with theadministration of a diet deficient in iron to treat or prevent the onsetof PCP in mammals. The amount of iron in the diet required to producesuch a deficiency in a mammal will, of course, vary according to thesubject but such amounts are ascertainable by those skilled in the art.

This invention further provides a method of treating or preventing theonset of Pneumocystis carinii pneumonia which comprises administering toa mammal in need of such treatment or prevention a compositioncomprising the following components: (a) a pharmaceutically acceptableiron chelator; (b) a compound selected fromtrimethoprim/sulfamethoxazole, pentamidine or other agent or combinationof agents known to be active against PCP; and (c) a pharmaceuticallycompatible carrier or diluent, wherein the amounts of said component (a)and said component (b) in combination are effective in treating orpreventing said pneumonia. Examples of iron chelators, and amounts ordosages, including such preferred amounts and dosages of iron chelators,TMP/SMZ, pentamidine, modes of administration and pharmaceuticallycompatible carriers or diluents have been described above. It should beunderstood that the amount of either component (a) or component (b)alone, might or might not be effective in treating or preventing saidpneumonia. The combination of the two components, (a) and (b), however,is effective for such treatment or prevention.

The present invention concerns a composition for the treatment orprevention of Pneumocystis carinii pneumonia in mammals comprising: (a)an amount of a pharmaceutically acceptable iron chelator; (b) an amountof a compound selected from trimethoprim/sulfamethoxazole (TMP/SMZ) andpentamidine, and a pharmaceutically compatible carrier, such as water,including, sterile or deionized Water, physiological saline, sodiumbicarbonate, and glucose.

This invention further concerns a composition for the treatment orprevention of Pneumocystis carinii pneumonia in mammals comprising:

(a) an amount of a pharmaceutically acceptable hydroxamic acid;

(b) an amount of pentamidine; and

(c) a pharmaceutically compatible carrier; wherein the total amounts ofsaid components (a) and (b) in combination are effective in treating orpreventing said pneumonia.

The iron chelators (a) in this composition include without limitationhydroxamic acids, preferably,N-[5-[3-[(5-aminopentyl)hydroxycarbamoyl]propionamido]pentyl]-3-[[5-(N-hydroxyacetamido)pentyl]carbamoyl]propionohydroxamicacid monomethane sulfonate (DFO), catechols, catecholates, pyridones,thiosemicarbazones and hydrazones and combinations of any of theforegoing that have the property to chelate iron. Effective amounts ofthe iron chelator may range from about 1 to about 260 mg/kg body weightper day, preferably from about 5 to about 100 mg/kg body weight, perday. When DFO is employed, the effective amount may range from about 0.2to about 6 g daily, preferably from about 0.5 to about 2.0 g daily.

The amount of the compound (b) in the composition, e.g., a dihydrofolatereductase inhibiting compound, e.g., TMP/SMZ or pentamidine, may varyfrom about 1 to about 100 mg/kg per day, i.e., from about 1 to about 20mg TMP/kg/day; from about 3 to about 100 mg SMZ/kg/day; and from about0.1 to about 2.0 mg pentamidine/kg/day. Those skilled in the art willappreciate that the amount of TMP/SMZ or pentamidine, as the case maybe, may be less than the effective amount of these reducing the toxicside-effects associated with their administration.

The components, i.e., iron chelator and compound selected from TMP/SMZand pentamidine, may be administered in accordance with the presentinvention in formulations such as liquids, e.g., suspensions,dispersions, which include sterile aqueous solutions or dispersions, orsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersions. The formulations may also be administered astablets, which are also available commercially, e.g., sulfamethoxazolewith trimethoprim tablets 400 mg w/80 mg, are available from BarrLaboratories, Inc., Northvale, N.J. 07647.

In the case of desferrioxamine (DFO), particularly the mesylate salt,the compound may be prepared for parenteral, i.e., intramuscular,intravenous or subcutaneous administration by dissolving in sterilewater and making sure that the drug is completely dissolved beforeadministering.

The use of iron chelators in the treatment of PCP may also yieldadditional unexpected advantages. It has been shown for example, thatDFO decreases collagen accumulation and lessens the severity of lungfibrosis induced by the antineoplastic agent bleomycin (Chandler, D.B.and Fulmer, J. D., Am. Rev. Respir. Dis., 131: 596-598, 1984).

The present invention is further described below in specific workingexamples which are intended to illustrate the invention without limitingthe scope thereof.

EXAMPLE 1 Treatment of PCP with DFO in a Rat Model for the Disease

Two examples of the efficacy of iron chelators in the treatment of PCPin a rat model are presented. These examples use DFO as a modelcompound. For the two examples, slightly different protocols were usedfor inducing immunosuppression in the rats and thus producing PCP.

The effects of DFO on rats suffering from PCP was examined as follows.To initiate immunosuppression, and continuing for the entireexperimental period, rats (5 per group) were given twice Weeklysubcutaneous injections of cortisone acetate (25 mg/per injectionCortone®, Merck Sharpe and Dohme, West Point, Pa.). To ensure completeimmunosuppression, the rats were maintained on a low protein diet (8.5%protein, W. F. Fisher and Sons, Bound Brook, N.J.) for the first 14 daysof the experiment. All food, bedding, cages and water bottles weresterilized before use. From the initiation of immunosuppression and forthe entire experimental period, protection from other infectious agentswas provided by the inclusion of 3.56 g (oxytetracycline HCl, Polyotic®Pfizer, Agricultural Division, New York, N.Y.) per liter of drinkingwater and by subcutaneous injection of 2.5 mg amphotericin B(Fungizone®, E. R. Squibb and Sons, Inc. Princeton, N.J.).) per animaltwice weekly. The initial weight of the rats was about 180 g but thisdeclined by about 35% during the course of the experiments.

Desferrioxamine mesylate (DFO, desferrioxamine, Desferral®, CIBAPharmaceutical Co., Summit, N.J.) was administered at the indicated doseby subcutaneous injection after reconstitution with 2.0 ml sterile waterper vial, i.e., according to the manufacturer's instructions. The entiredaily dose was given in a single injection.

The intensity of the P. carinii infection was judged from smears madefrom homogenates of rat lung. This procedure involved the removal ofboth lungs from a rat and then pressing the lungs through a stainlesssteel garlic press. The extruded lung tissue was then suspended in 5.0ml of a buffer comprising 0.1M Na₂ HPO₄ (Sigma Chemical Co., St. Louis,Mo.), 0.073M NaCl (Sigma Chemical Co., St. Louis, Mo.), 100 mMethylenediaminetetraacetate (Mallinckrodt Inc., St. Louis, Mo.), pH 7.4.The suspended lung tissue was then forced through a stainless steel teastrainer with the aid of the plunger from a 5-ml disposable syringe(Becton Dickinson and Co., Rutherford, N.J.). A smear of the homogenatewas prepared and stained with cresyl echt violet (Roboz SurgicalInstrument Corp., Washington, D.C.) to reveal Pneumocystis carinii cysts(as described in Bowling et al., Am. J. Med. Technol., 39:267-268,1973). Special attention was paid to maintain the pH of the stain at1.5. The process of staining for P. carinii cysts involved first airdrying then heat fixing the smear. They were then held 10 minutes in acoplin jar (Fisher Scientific, Springfield, N.J.) containing 60 mlglacial acetic acid (Fisher Scientific) and 20 ml concentrated sulfuricacid (Fisher Scientific) and frequently agitated. The slides were rinsedin tap water for 8 minutes and placed for 25 minutes in another coplinjar containing the staining solution made of 0.1 g cresyl echt violet,60 ml 0.1N HCl and 40 ml 0.1M NaH₂ SO₄. The staining solution had beenmade at least 24 hours before use, filtered through fluted filter paper(Whatman 2 V, Fisher Scientific) and the pH adjusted to 1.5. The slidewas then rinsed in tap water and placed for 2 minutes in a coplin jarcontaining 0.01 g naphthol yellow (Aldrich Chemical Co., Milwaukee,Wis.) and 100 ml of 1% glacial acetic acid. The slides were rinsed,air-dried and examined for cysts with a 100× oil immersion lens. Thecysts in 250 fields were counted and this count was the PCP intensityscore.

Experimental therapies were begun 62 days after initiation ofimmunosuppression. The experimental therapies were given for 3 weeks andthe animals were then sacrificed and examined for P. carinii in thelungs.

                  TABLE 1                                                         ______________________________________                                        EVALUATION OF DESFERRIOXAMINE                                                 IN A RAT MODEL OF Pneumocystis carinii PNEUMONIA                                                     Individual                                                        Mean        Animal      Mean                                                  Daily       Infections  Infection                                             Drug        After 3 Weeks                                                                             ± SEM                                              Dosage      (cysts/250  (cysts/250                                 Treatment  (mg/kg-1 day)                                                                             fields)     fields)                                    ______________________________________                                        None       N/A*        37          96.6 ± 48.3                             (Negative              74                                                     Control)               287                                                                           27                                                                            58                                                     Trimethoprim                                                                             26.2         0                                                     +          +            0          0 ± 0                                   Sulfamethoxazole                                                                         130.9        0                                                     in                      0                                                     Drinking Water          0                                                     (Positive Control)                                                            Desferrioxamine                                                                          1000        13          15.0 ± 5.3                              s.c.                   34                                                                            17                                                                             7                                                                             4                                                     ______________________________________                                         * = Not Applicable                                                       

As can be seen from the data in Table I, while the TMP/SMZ treatment washighly effective, DFO suppressed PCP by an average of 85% in the ratmodel. Therefore, DFO provided a substantial reduction in the P. cariniipopulation without the side effects attendant with TMP/SMZ treatment andthus permits a viable alternative (or adjunct) to TMP/SMZ therapy.

EXAMPLE 2

The data for Example 2 are presented in Table 2. The immunosuppressionprotocol for Example 2 was the same as in Example 1 except the lowprotein diet was maintained for the entire experimental period, noamphotericin B was administered, and infection was initiated solely byexposure via the room air to other animals which had been placed onimmunosuppression earlier.

The effect of a range of DFO dosages is presented in Table 2.

                  TABLE 2                                                         ______________________________________                                        FURTHER EVALUATION OF DESFERRIOXAMINE                                         IN A RAT MODEL OF Pneumocystis carinii PNEUMONIA                                                    Daily     Individual Mean                                                     Animal    Cyst Count                                                Drug      Cyst Count                                                                              +SEM                                                      Dosage    (cysts/250                                                                              (cysts/250                                    Treatment   (mg kg-1) fields)   fields)                                       ______________________________________                                        None                   0        1451 ± 762                                 (Negative Control)    6250                                                                          123                                                                           37                                                                            4500                                                                          609                                                                           104                                                                           850                                                                           589                                                     desferrioxamine                                                                           1000      13        9 ± 3                                                            15                                                                            20                                                                             0                                                                            10                                                                             4                                                                             5                                                                             2                                                      desferrioxamine                                                                           500       16        27 ± 10                                                          77                                                                             7                                                                             0                                                                            27                                                                            20                                                                            60                                                                             8                                                      desferrioxamine                                                                           250       179       106 ± 46                                                         95                                                                            71                                                                             4                                                                             4                                                                            48                                                                            343                                                     ______________________________________                                    

From the data shown in Table 2 above, it can be seen that DFOsignificantly inhibited cyst formation in the lungs of treated animalsat all dosages (250-1000 mg kg⁻¹).

EXAMPLE 3

In this experiment, a pyridone iron chelator,1,2-dimethyl-3-hydroxypyrid-4-one (DMHP) (Kontoghiorghes, G. J. andSheppard, L., Supra) is administered to an immunosuppressed rat model.The rat subjects are immunosuppressed by following the immunosuppressionprotocol of Example 2. DMHP is administered orally or intrapentoneallyto rate subjects in the following amounts (dosages): 10, 20, 40, 75,150, 300 and 600 mg/kg/day. DHMP is prepared as the hydrochloride saltby dissolving in water with the total volume administered to the ratsubjects being 1 ml/100 g body weight. The evaluation of the efficacy ofDMHP is carried out according to the evaluation set forth in Example 2.

What is claimed is:
 1. A method of treating pneumonia caused byPneumocystis carinii comprising administering (a) a pharmaceuticallyacceptable iron chelator and (b) pentamidine wherein the amounts of (a)and (b) in combination are effective in treating pneumonia caused byPneumocystis carinii.
 2. A method of treating pneumonia caused byPneumocystis carinii comprising administering (a) pentamidine and (b) ahydroxamic acid wherein the amounts of (a) and (b) in combination areeffective in treating pneumonia caused by Pneumocystis carinii.
 3. Amethod of preventing the onset of pneumonia caused by Pneumocystiscarinii comprising administering (a) a pharmaceutically acceptable ironchelator and (b) pentamidine wherein the amounts of (a) and (b) incombination are effective in preventing pneumonia caused by Pneumocystiscarinii.
 4. A method of preventing the onset of pneumonia caused byPneumocystis carinii comprising administering (a) pentamidine and (b) ahydroxamic acid wherein the amounts of (a) and (b) in combination areeffective in preventing pneumonia caused by Pneumocystis carinii.
 5. Acomposition for the treatment or prevention of pneumonia caused byPneumocystis carinii in mammals comprising:(a) an amount of apharmaceutically acceptable hydroxamic acid; (b) an amount ofpentamidine; and (c) a pharmaceutically compatible carrier, wherein thetotal amounts of said components (a) and (b) in combination areeffective in treating or preventing said pneumonia.